Fabricated copper catalyst on biochar nanoparticles for the synthesis of tetrazoles as antimicrobial agents

Article abstract of DOI:10.1016/j.poly.2019.114169

Biochar is a carbon-rich solid which its surface was covered by high density of carbonyl, hydroxyl and carboxylic acid functional groups. In this work, biochar nanoparticles were prepared from pyrolysis of chicken manure and further a new copper catalyst

Full text of DOI:10.1016/j.poly.2019.114169

Journal Pre-proofs
Fabricated copper catalyst on biochar nanoparticles for the synthesis of tetra-
zoles as antimicrobial agents
Parisa Moradi, Maryam Hajjami, Bahman Tahmasbi
PII:
S0277-5387(19)30606-0
DOI:
https://doi.org/10.1016/j.poly.2019.114169
POLY 114169
Reference:
To appear in:
Polyhedron
Received Date:
Revised Date:
Accepted Date:
9 June 2019
24 September 2019
4 October 2019
Please cite this article as: P. Moradi, M. Hajjami, B. Tahmasbi, Fabricated copper catalyst on biochar nanoparticles
for the synthesis of tetrazoles as antimicrobial agents, Polyhedron (2019), doi: https://doi.org/10.1016/j.poly.
2
019.114169
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Fabricated copper catalyst on biochar nanoparticles for the synthesis of
tetrazoles as antimicrobial agents

Parisa Moradi, Maryam Hajjami , Bahman Tahmasbi
Department of Chemistry, Faculty of Science, Ilam University, P. O. Box 69315516, Ilam, Iran.
Abstract:
Biochar is a carbon-rich solid which its surface was covered by high density of carbonyl,
hydroxyl and carboxylic acid functional groups. In this work, biochar nanoparticles were
prepared from pyrolysis of chicken manure and further a new copper catalyst was supported on
its surface (Cu-ABA@biochar) as reusable and environmental biocatalyst. The structure of this
catalyst has been characterized by SEM, EDS, WDX, XRD, TGA, N adsorption–desorption
2
isotherms, FT-IR and AAS techniques. SEM images of this catalyst demonstrate that particle
size is less than 100 nm in diameter. The catalytic activity of this nanocatalyst was studied in the
synthesis of tetrazole derivatives. Tetrazole products were obtained in high TOF and TON values
in the presence of Cu-ABA@biochar, which indicate the high efficiency of this catalyst. The
biological activity of obtained tetrazoles was screened against several Gram-positive & Gram-
negative bacteria strains which these compounds were found to possess promising activity. This
catalyst was reused for several runs without significant loss of its catalytic activity or copper
leaching. Also, recovered catalyst was characterized by SEM, FT-IR, AAS and N adsorption–
2
desorption isotherms. Heterogeneity and stability of this nanocatalyst was confirmed by hot
filtration test and reusing of catalyst.
Key words: Biochar Nanoparticles, Copper, Tetrazoles, Biocatalyst, Antimicrobial.
Address correspondence to Maryam Hajjami, Department of Chemistry, Faculty of Science, Ilam University, P.O. Box 69315516,
Ilam, Iran; Tel/Fax: +98 843 2227022; E-mail address: mhajjami@yahoo.com or m.hajjami@ilam.ac.ir
1

1
. Introduction
Nanoparticles were emerged in various fields especially in catalysis application which they are
used as catalyst directly or were used as support for heterogenization of catalysts [1-4].
Insolubility, remarkable efficiency, stability and high surface area of nanoparticles provide a
suitable properties for their catalytic application [5-8]. Therefore, nanomaterials can applied as a
bridge gap between heterogeneous and homogeneous catalyst [7]. In this regard, several
nanomaterials were developed in the last few decades for extension of catalysis science. Among
them, metal oxide nanoparticles [9], mesoporous materials [10, 11], polymers [12], graphen
oxide [13], ionic liquids [14], boehmite [15, 16], MOF nanoparticles [17, 18] and carbon
nanostructures [19-21] were widely used as catalyst or support. These known nanoparticles have
various advantage and disadvantages meanwhile, several disadvantage are common in all of
them. For example, preparation of these nanomaterials need to chemical starting materials which
led to expensive procedure and also preparation of these nanomaterials is combine to generation
of chemical wastes which not environmentally friendly. Therefore we introduced biochar as new
support because biochar was made via pyrolysis of biological source such as agricultural wastes,
green waste, woody materials, animal manures and other waste products [22-24], therefore it is
inexpensive and environmentally friendly. In fact, biochar is called as black carbon which is a
stable solid in air atmosphere, high temperature and aqueous solution, and was used as a soil
amendment, reduction in greenhouse gas emissions from soil, agricultural waste recycling, water
retention, adsorption, and climate change mitigation [23-27]. Despite, existence of several
studies on biochar properties, or its application as soil amendment [27-29], biochar has been
rarely used as catalyst or heterogeneous support for the immobilization of homogeneous catalysts
2

[22, 30, 31]. High density of carboxylic acid, carbonyl and hydroxyl groups on the surface of
biochar [22, 27, 28] provide to modification of its surface for immobilization of catalysts. In this
regards, we are reporting a new complex of copper on biochar nanoparticles (Cu-TBA@biochar)
as efficient and reusable biocatalyst for synthesis of 5-substituted tetrazoles.
Tetrazoles are great class of five-membered and nitrogen-rich heterocyclic compounds, which
have been reported as analgesic, anti-proliferative, anti-inflammatory, antibacterial, antiviral,
antifungal, potential anti HIV drug candidates, herbicidal and anticancer agents [32-41]. For
example olmesartan medoxomil, TAK-456, losartan and valsartan are several compounds
including tetrazole core with drugs applications such as antihypertensive family drugs [33-35].
Also, tetrazoles are used in coordination chemistry as ligands and were employed as stable
surrogates for carboxylic acids [38, 42]. [3 + 2] cycloaddition of azide derivatives to the nitriles
is conventional method for the synthesis of 5-substituted 1H-tetrazoles [42-46].
2
. Experimental
2
.1. Materials and instruments
Chemicals and solvents in this work were purchased from Merck, Aldrich, or Fluka companies
and used without further purification. Powder XRD of biochar nanoparticles and obtained
catalyst were performed with Cu Kα radiation at 40 kV and 30 mA using a PW1730 instrument
from Philips Company. Thermogravimetric analyses (TGA) of the catalyst was recorded by a
SDT Q600 V20.9 Build 20 device in the temperature range of 30–800 °C under air atmosphere.
The particle size and morphology of prepared nanoparticles were studied by SEM images using
FESEM-TESCAN MIRA3 and FEI Quanta 200 Scanning Electron Microscopes, on an
accelerating voltage of 25.0-30.0 kV. FT-IR spectra were recorded with KBr pellets using a
3

VRTEX 70 model Bruker FT-IR spectrometer. The component analysis was carried out by the
energy dispersive X-ray spectroscopy (EDS) using an FESEM-TESCAN MIRA3 Scanning
Electron Microscope. Nitrogen adsorption isotherms were obtained using a standard gas
manifold at 77 K to surface characteristics of the catalyst using a BELSORP MINI II device.
Also, the catalyst sample was degassed at 120 °C for 2 h using a BEL PREP VAC II device
before analysis. The exact content of copper was measured by AAS technique using 400p-
novAA instrument from Analytik Jena Company. CHNSO analysis of biochar was performed
using ECS 4010 Element Analyzer from COSTECH Company. NMR spectra of products were
recorded using Bruker DRX-400 spectrometer at 400 MHz.
2
.2. Preparation of biochar
5
00 g of dried chicken manure was placed in a porcelain crucible. The pyrolysis temperature was
selected from 400 °C to 800 °C, which is the common temperature range of pyrolysis. The set
temperature reached after about 30 min of heating with the carrier gas (N ) sweeping at 0.3
2
-1
L.min , and the porcelain crucibles were fed into the heating zone with an N flow rate of 0.03
2
-1
L.min . After 1 and 2 h, the pyrolysis process was ended, and the porcelain crucibles were
-1
removed from the heating zone and cooled with N sweeping at 0.3 L.min for 30 min. The solid
2
yield was the biochar, which was ground through a 40 mesh sieve (0.45 mm). Prior to additional
experiments, no pretreatment was performed. The obtained biochar was abbreviated as
CMB400-1, CMB400-2 CMB600-1, CMB600-2 and CMB800-1, CMB800-2, respectively
according to the pyrolysis residence time (1 and 2 h) and temperature. Carbon content, ash
Content and char yield of biochar are shown in Table 1. Also, the element content of obtained
biochar was studied by CHNSO analysis and the obtained results are summarized in Table 2.
4

Table 1. Typical char yield, ash Content and carbon content of obtained biochar
Entry
Sample
Char yield (wt.%)
Carbon content
Ash Content (%)
1
2
3
4
5
6
CMB 400-1
CMB 400-2
CMB 600-1
CMB 600-2
CMB 800-1
CMB 800-2
45.3
37.45
14.25
12.34
10.52
8.54
40.4
36.36
11.8
8.9
1.5
50
60
80
90
90
90
1.06
Table 2. Typical char yield, ash Content and carbon content of obtained biochar
Elemental analysis
CMB 400-1
CMB 400-2
CMB 600-1
CMB 600-2
CMB 800-1
CMB 800-2
C
H
N
O
S
-
-
40.4
36.36
11.8
8.9
1.5
1.06
1.8
4.2
24.24
21.81
4.72 0.14
3.5
0.3
0.21 0.15
1.62
0.42
0.32
0.06
0.15
3.69
1.15
0.68
0.09
0.02
-
-
2
.3. Preparation of catalyst
Initially, 2-(thiophen-2-yl)-1H-benzo[d]imidazole (TBA) as ligand for immobilization of Copper
has been synthesized according to reported procedure by Nagawade et. al. (Scheme 3) [47].
NH2
N
ZrOCl (10 mol%)
2
OHC
+
S
N
H
S
NH2
EtOH, r.t., 2.5 h
Scheme 1. Synthesis of 2-(thiophen-2-yl)-1H-benzo[d]imidazole (TBA).
Biochar nanoparticles modified by 3-choloropropyltrimthoxy silane (CPTMS@biochar)
according to new reported procedure [48]; subsequently CPTMS@biochar (1.0 g) was dispersed
in toluene and 1.5 mmol of TBA was added to this mixture. The mixture was stirred at 90 ºC for
4
8 h. The solid product (TBA@biochar) was isolated after washing with ethanol and drying at 50
5

˚C. The obtained TBA@biochar (0.5 g) was dispersed in 25 mL ethanol by sonication for 20
min, and then Cu(NO ) .9H O (1 mmol) was added to the reaction mixture. The reaction mixture
3
2
2
was stirred at 80 ºC for 20 h. The final product (Cu-TBA@biochar) was filtered, washed by
ethanol/water and dried at room temperature.
2
.4. General procedure for the synthesis of 5-substituted 1H-tetrazoles in the presence of Cu-
TBA@biochar
A mixture of NaN (1.7 mmol) and benzonitrile derivative (1 mmol) in the presence of 0.050 g
3
of Cu-TBA@biochar, were stirred at 130 °C in PEG-400; after completion of the reaction
(observed by TLC), the reaction mixture was cooled down, and reaction mixture was diluted by
water and ethyl acetate. Then, the catalyst was isolated by simple filtration. The remained
catalyst was washed with water and ethy acete for several times. Then, aqouse solution of HCl (4
N, 10 mL) was added to the filtrated solution. The pure products extracted with ethyl acetate
from water. The organic solvent was dried over anhydrous sodium sulfate, and concentrated to
give the crude solid product. High purity of products was achieved using thin layer
chromatography in a mixture of n-hexan and aceton as mobile phase.
2
.5. Selected spectral data
-(4-bromophenyl)-1H-tetrazole: H NMR (400 MHz, DMSO): δ = 17.05 (b, 1H), 8.01-7.99
1
5
H
(d, J= 8 Hz, 2H), 7.86-7.84 (d, J= 8 Hz, 2H) ppm.
1
5
-(4-chlorophenyl)-1H-tetrazole: H NMR (400 MHz, DMSO): δ = 8.08-8.06 (d, J= 8 Hz,
H
2
H), 7.72-7.70 (d, J= 8 Hz, 2H) ppm.
1
1
-(4-(1H-tetrazol-5-yl)phenyl)ethanone: H NMR (400 MHz, DMSO): δ = 8.22-8.19 (d, J= 12
H
Hz, 2H), 8.19-8.16 (d, J= 12 Hz, 2H), 2.66 (s, 3H) ppm.
1
4
-(1H-tetrazol-5-yl)benzonitrile: H NMR (400 MHz, DMSO): δ = 8.24-8.22 (d, J= 8 Hz, 2H),
H
8
.11-8.02 (d, J= 8 Hz, 2H) ppm.
6

1
5
-phenyl-1H-tetrazole: H NMR (400 MHz, DMSO): δ = 8.07-8.05 (t, J= 8 Hz, 2H), 7.65-7.59
H
(m, 3H) ppm.
1
2
-(1H-tetrazol-5-yl)benzonitrile: H NMR (400 MHz, DMSO): δ = 8.15-8.09 (m, 2H), 7.99-
H
7
.94 (m, 1H), 7.84-7.81 (m, 1H) ppm.
. Result and discussion
3
3
.1. Catalyst preparation
In this research project, at first Cu-TBA@biochar was prepared which the procedure for the
synthesis of this catalyst is outline in Scheme 1. Then Cu-TBA@biochar was characterized by
scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), X-ray
diffraction (XRD), thermogravimetric analysis (TGA), Fourier transform infrared spectroscopy
(FT-IR), Brunauer-Emmett-Teller (BET), wavelength dispersive X-ray spectroscopy (WDX),
and atomic absorption spectroscopy (AAS).
Cl
Cl
Si
OHC CHO
HOOC HO
3-CPTMS
n-hexane, reflux, 24 h
Si
O
O
O
O
HO
O
O
N
HO
OH
S
N
S
N
N
S
N
N
H
Si
O
Toluene, 90 °C, 48 h
Si
O
O
O
O
O
Cu
N
S
Cu
S
N
N
N
Cu(NO) .9H O
3
2
Si
O
Si
O
O
EtOH, 80 °C, 20 h
O
O
O
Scheme 1. Synthesis of Cu-TBA@biochar.
7

3
.2. Catalyst characterizations
The SEM technique was used to studying of morphology and size of particles in free biochar and
catalyst. The SEM image of free biochar was compared with SEM image of Cu-TBA@biochar.
As shown in Figure 1, the particle of biochar is quasi-spherical with an average diameter about
3
0-70 nm which are good agreement to shape and size of Cu-TBA@biochar. These results were
confirmed that particles of biochar were not any change after surface modification or
immobilization of catalyst.
a
b
Figure 1. SEM images of (a) biochar and (b) Cu-TBA@biochar
EDS analysis was performed to determine the elements content of Cu-TBA@biochar (Figure 2)
and also WDX analysis was performed to determine the elements distributions of Cu-
TBA@biochar (Figure 3). As depicted, the EDS results of Cu-TBA@biochar indicate elements
8

content of Cu-TBA@biochar as oxygen, carbon, silica, sulfur, nitrogen, and as well as copper
species in the structure of the catalyst. Also, Figure 3 was shown the WDX analysis of Cu-
TBA@biochar. As shown in Figure 3, the homogeneous distribution of oxygen, carbon, silica,
sulfur, nitrogen, and copper was clearly observed in WDX analysis from Cu-TBA@biochar.
Also, the exact amount of copper in the structure of Cu-TBA@biochar was evaluated by AAS
-3
-1
analysis that found to be 0.78 × 10 mol g .
.
Figure 2. EDS diagram of Cu-TBA@biochar.
9

a
b
c
d
e
f
Figure 3. Elemental mapping of (a) carbon, (b) nitrogen, (c) oxygen, (d) silica, (e) sulfur, and (f) copper for Cu-
TBA@biochar.
TGA diagram of Cu-TBA@biochar is shown in Figure 4. This diagram shows a small mass loss
about 4% in the low temperature which is lower than 150 °C. This weight loss related to the
evaporation and removal of adsorbed solvents [38]. The TGA diagram of the Cu-TBA@biochar
was shown a mass loss between 300-600 °C which is about 50 %. This mass loss can be related
to the organic content in catalyst [14]. As shown in Figure 4, no weight loss was not observed
between 150-300 °C which is indicate the stability of catalyst up to 300 °C. Therefore this
1
0

catalyst can be applied for organic reaction in high temperatures and hard conditions. Based on
these results, an interesting advantage of Cu-TBA@biochar as catalyst is high thermal stability
which allowed that application of this catalyst can be extended for wide ranges of organic
reactions.
Figure 4. TGA diagram of Cu-TBA@biochar.
The normal XRD patterns of free biochar and Cu-TBA@biochar are shown in Figure 5. A sharp
peaks at 2θ°=30° were shown in the XRD patterns of biochar and Cu-TBA@biochar which is
agreement to standard XRD pattern of biochar [31, 49]. Figure 5 also shows several weak peaks
at 2θ°=40.7°, 43.7°, 48.7° and 66.7° for XRD patterns of biochar and catalyst [49]. As shown in
Figure 5, XRD pattern of biochar shows a good agreement with XRD pattern after modification.
1
1

Figure 5. Normal XRD pattern of biochar (a) and Cu-TBA@biochar (b).
3
.3. Catalytic application of Cu-TBA@biochar in the synthesis of tetrazoles
The catalytic activity of Cu-TBA@biochar was studied in the synthesis of tetrazole derivatives.
Synthesis of tetrazole derivatives is shown in Scheme 2. In the first step, [3 + 2] cycloaddition of
sodium azide and benzonitrile was selected as model reaction to optimization of reaction
conditions. This model reaction was examined in various parameters such as different amount of
catalyst, several solvent and wide range of temperatures. The obtained results from these
experiments are summarized in Table 3. As shown in Table 3 (Entry 5), the best results
(including product yield and reaction time) were obtained in the presence of 50 mg (containing
0
.78 mol%) of Cu-TBA@biochar. Decreasing in the amount of catalyst from 50 to 40 mg was
led to decreasing in yield of product and increasing in reaction time (Table 3, entry 2). Also,
among the various solvents, the best results were obtained in PEG-400. When PEG-400 was used
as solvent, 98% of products were obtained after 7 h. while, when H O, DMSO, DMF and ethanol
2
were used as solvent, 59%, 43%, 30% and 38% of products were obtained respectively after 7 h.
1
2

Therefore, PEG-400 was selected as solvent. Finally, the wide ranges of temperatures (between
room temperature-130 °C) were also studied and the best results were obtained at 130 °C.
N
Cu
N
O
Si
S
O
O
CN
N
Cu-TBA@biochar
PEG-400, 130 °C
N
N
NaN3
+
R
N
H
R
Scheme 2. Synthesis of tetrazoles in the presence of Cu-TBA@biochar
Table 3. Optimization reaction conditions for the synthesis of tetrazoles in the presence of Cu-TBA@biochar.
Yield
Entry Catalyst (mg)
Solvent
Temperature (ºC)
Time (h)
a
(%)
1
2
3
4
5
6
7
8
9
-
PEG
PEG
PEG
PEG
PEG
120
120
120
120
130
130
130
Reflux
Reflux
r.t
24
22
20
17
7
7
7
7
7
N.R
84
95
93
98
43
30
59
38
40
50
60
50
50
50
50
50
50
DMSO
DMF
H O
2
EtOH
PEG
1
0
7
N.R
a
Isolated yield.
The catalytic activity of Cu-TBA@biochar was explored in [3 + 2] cycloaddition of
sodium azide with various benzonitrile derivatives (Table 4). Aliphatic and aromatic
nitriles bearing electron-donating or electron-withdrawing groups were studied and
corresponding tetrazoles were obtained in high yields and high TOF values. High TON
1
3

and TOF values were indicate the high efficiency of Cu-TBA@biochar in the synthesis of
tetrazole derivatives. Interestingly, dicyano substituted derivatives such as phthalonitrile
(Table 4, entry 11), terephthalonitrile (Table 4, entry 7), malononitrile (Table 4, entry 16)
and 2‐benzylidenemalononitrile derivatives (Table 4, entry 10) were afforded the mono
addition products. Therefore this procedure can be explored for wide ranges of nitriles.
Table 4 Synthesis of tetrazole derivatives in the presence of Cu-TBA@biochar.
Time
Yield
(%)a
-1
Entry
Nitrile
Product
TON TOF (h )
(min)
N
N
CN
N
1
N
420
98
95
125.6
121.8
18.4
10.7
H
N
N
CN
N
2
3
4
N
H
680
15
Br
Br
N
N
CN
OH
N
N
N
N
96
96
123.1
123.1
492.4
98.5
H
OH
N
N
CN
Cl
N
75
H
Cl
N
N
CN
N
H
5
405
98
125.6
18.6
Cl
Cl
1
4

N
N
CN
N
N
6
H
530
92
117.9
13.3
O
NC
Cl
O
NC
Cl
N
N
N
N
CN
CN
CN
N
7
8
9
N
H
140
140
75
97
89
91
124.3
114.1
116.7
53.3
48.9
93.4
N
N
N
H
N
N
N
H
HO
HO
N
N
CN
CN
N
N
1
0
H
95
93
119.2
75.3
CN
N
O N
O N
2
2
N
CN
CN
N
1
1
1
2
N
70
91
88
116.7
112.8
100
H
CN
N
N
CN
N
N
300
22.6
H
O N
2
O N
2
N
N
CN
N
N
H
270
95
121.8
27.1
1
3
NO2
NO2
1
5

N
1
1
1
4
5
6
N
N
53
120
25
90
85
96
115.4
109
130.6
54.5
CN
HN
N
O
N
O
N
CN
O
N
O
H
N
NC
N
123.1
295.4
NC
CN
HN
N
a
Isolated yield after purification.
3
.4. Antimicrobial activity tetrazoles
The antibacterial activity of 5-(3-nitrophenyl)-1H-tetrazole (Table 4, entry 13) and 4-(1H-
tetrazol-5-yl)phenol (Table 4, entry 9) was evaluated by determination of their minimum
inhibitory concentrations (MICs) by using agar dilution method. For this regard, growth medium
of Mueller-Hinton agar was used for antibiotic susceptibility testing. The inhibitory effects of
this compounds was studied against bacterial strains which comprise of three Gram negative
bacteria and three Gram positive bacteria. Escherichia coli (ATCC 25922), Klebsiella
pneumoniae (ATCC 25926) and Pseudomonas aeroginosa (ATCC 27853) were selected as Gram
negative bacteria, also Bacillus cereus (ATCC 6177), Staphylococcus aureuse (ATCC 25923)
and Streptococcus agalactiae (ATCC 12232) were selected as Gram positive bacteria. The MIC
values are compared with standard drugs Tetracycline (30 mg/mL) and Gentamicin (10 mg/mL).
At a glimpse, 5-(3-nitrophenyl)-1H-tetrazole (13a) has no antibacterial effect. Meanwhile, 4-
(1H-tetrazol-5-yl)phenol (9a) showed significant antibacterial activity against of both Gram
negative and Gram positive bacteria. Compound 9a showed a good inhibitory efficiency against
of Escherichia coli (MIC 10 mg/mL), Klebsiella pneumoniae (MIC 5 mg/mL) and
1
6

Staphylococcus aureuse (MIC 10 mg/mL). 5 mg/mL of 9a has a lower inhibitory effect against
Klebsiella pneumoniae than 10 mg/mL of it.
3
.5. Recyclability of the catalyst
The recovering and recycling of the Cu-TBA@biochar were studied in the synthesis of 5-(2-
chlorophenyl)-1H-tetrazole using [3 + 2] cycloaddition of sodium azide with 2-
chlorobenzonitrile as model reaction. In this regard, after completion of each reaction, reaction
mixture was diluted by ethyl acetate and water. Then, the catalyst was recovered by
centrifugation. The remained catalyst was washed with ethy acete and water for several times.
Then, aqouse solution of HCl (4 N, 10 mL) was added to the filtrated solution. 5-(2-
chlorophenyl)-1H-tetrazole as product extracted with ethyl acetate from water. The recovered
catalyst was reused in the next run. The obtained results from recycling of Cu-TBA@biochar
are summarized in Figure 6. As shown in Figure 6, this catalyst can be recycled up to 8 times
without significant loss in its catalytic activity.
Figure 6. Recyclability of Cu-TBA@biochar in synthesis of 5-(2-chlorophenyl)-1H-tetrazole.
1
7

3
.6. Leaching Study of catalyst
In order to studying of metal leaching in the reaction mixture, hot filtration test was performed.
For this regard, [3 + 2] cycloaddition of sodium azide with benzonitrile was selected and further
in half‐time of the reaction (the half‐time of this reaction is 210 minutes), the product was
isolated which 47% of product was obtained. Then, this reaction was repeated, meanwhile after
half‐time of reaction, catalyst was remover and then permitted to continue reaction for the
filtrated solution. In this stage, after 7 h the product was obtained in 52% of yield. These
experiments confirm that leaching of copper did not occur.
In order to explore the studding about of heterogeneous nature of Cu-TBA@biochar, copper
concentration in reaction solution was calculated by AAS analysis. In this regard, reaction of
sodium azide and benzonitrile was repeated under optimized conditions. Then, catalyst was
removed by filtration after 210 min and further the filtered solution was analyzed by AAS
technique. In this analysis, the copper concentration in solution was very small, which found to
-6
-1
be 6.7 × 10 mmol mL . This very small amount of cooper did not have a significant effect on
the reaction progress. Therefore completion of the reaction could be attributed to the supported
copper on biochar.
3
.7. Characterization of recycled catalyst
In order to indicate the stability of Cu-TBA@biochar after recovering and reusing, recovered
catalyst was characterized by SEM, BET, AAS and FT-IR techniques. The morphology and
particle size of Cu-TBA@biochar after recycling has been studied by SEM technique. The SEM
image of reused Cu-TBA@biochar is shown in Figure 7. As shown in this Figure, the size and
morphology of catalyst after reusing is shown a good agreement to fresh catalyst, which is about
1
8

3
0–70 nm in diameters . also, the shape of recycled catalyst was shown a good similarly to fresh
catalyst. In addition, AAS analysis of reaction solotion confirmed that copper leaching was not
happend under reaction conditions, which proves that Cu-TBA@biochar has heterogeneous
nature.
Figure 7. SEM images of recovered Cu-TBA@biochar
Nitrogen adsorption–desorption isotherms of recoverd and fresh catalyst are shown in Figure 8.
Also, textural properties of biochar, catalyst and recovered catalyst are summarized in Table 5.
Based on Brunauer-Emmett-Teller (BET), the BET surface area [31], pore volumes [31] and
pore diameters [31] of biochar are higher than fresh and recoverd catalyst which is due to the
grafting of organic layers and Cu-complex on the surface of biochar nanoparticles. Meanwhile, a
good agreement was observed between textural properties of recoverd and fresh catalyst.
1
9

Figure 8. Typical N adsorption–desorption isotherm of fresh (a) and recovered (b) Cu-
2
TBA@biochar
Table 5. Textural properties of biochar, fresh and recovered Cu-TBA@biochar.
2
3 -1
Entry
Sample
Biochar
Fresh Cu-TBA@biochar
Recovered Cu-TBA@biochar
SBET (m /g)
Pore diameter (nm) Pore volume (cm g )
1
2
3
199-557 [31]
16.4
2.4-3.0 [31]
1.29
0.03-0.22 [31]
0.08
14.4
1.29
0.05
FT-IR spectra of recovered catalyst was compared to FT-IR spectra of fresh catalyst which are
shown in Figure 9. As shown in Figure 9, good agreement was observed for FT-IR spectra of
fresh and recovered catalyst. Stability of Cu-TBA@biochar after recycling was confirmed with
position of stretching vibrations in FT-IR spectra of fresh and recycled catalyst which are
2
0

sammurized in Table 6. These results are strong evidence for stability of Cu-TBA@biochar in
reaction conditions.
Figure 9. FT-IR spectra of fresh (a) and recovered (b) Cu-TBA@biochar
Table 6. FT-IR characteristic absorption of fresh and recovered Cu-TBA@biochar.
-
1
Entry
Functional group
C–H stretch
C–H bending
C=C bending
O–H stretch
O–H bending
C=N stretch
C=O stretch
Si-O
Absorption (cm )
2858-2924
878
Reference
[5, 50]
[30]
[30, 31]
[16, 50]
[30, 31]
[7, 51]
[22, 31]
[52]
1
2
3
4
5
6
7
8
9
1562
3200-3650
1387
1632
1693
471, 795, 1075
1009-1111
710-795
C-O and C-N stretch
C-S stretch
[30, 31]
[53]
1
0
2
1

3
.8. Comparison of the catalyst
In order to shown the efficiency of Cu-TBA@biochar, this catalyst was compared with previous
methods in the [2+3] cycloaddition reaction of benzonitrile with sodium azide (Table 7). As
shown in Table 7, the TON and TOF values and also obtained yield in the presence of Cu-
TBA@biochar are higher than previous catalysts. Also biochar is inexpensive and
environmentally friendly in comparison with other supports because biochar is made via
pyrolysis of biological source such as agricultural wastes and green wastes. Also, many
previously reported methods were used of homogeneous catalysts which cannot recovered and
reused. While, Cu-TBA@biochar was recycled for several runs in synthesis of 5-(2-
chlorophenyl)-1H-tetrazole without significant loss of its catalytic activity, which demonstrates
the practical reusability and efficiency of this catalyst.
Table 3. Comparison of Cu-TBA@biochar in the synthesis of 5-phenyl-1H-tetrazole with previously reported
procedures.
-1
Entry
Catalyst
(NH )Ce(NO
Sm@l-MSN
@SiO /Salen Cu(II)
Condition
DMF, 110ºC
DMF, 100 ºC
DMF, 120ºC
Time (h) Yield (%)
TOF (h )
Ref.
[35]
[42]
[54]
1
2
3
4
3
)
6
6
5
7
97
87
90
1.61
40.7
-
Fe
3
O
4
2
ammonium chloride and
ammonium fluoride
2
DPOL/H O
4
48
95
0.10
[55]
(6/4), 160ºC
DMF, 120ºC
DMF, 120ºC
DMF, 110ºC
DMF, 120ºC
PEG-200,
5
6
7
8
AgNO
Cu(OAc)
CAN supported HY-zeolite
FeCl –SiO
3
5
12
4
83
98
93
79
1.66
0.32
0.77
-
[56]
[57]
[40]
[58]
2
3
2
12
9
Fe
3
O
4
@SiO
2
/aza-crown ether-Cu(ІІ)
2.5
7
95
98
38
[59]
1
00 ºC
This
work
1
0
Cu-TBA@biochar
PEG, 130ºC
18.4
2
2

4
. Conclusion
In conclusion, biochar was prepared from chicken manure and was used as support for
immobilization of copper as catalyst. At first this catalyst was characterized by SEM, EDS,
WDX, AAS, FT-IR, N adsorption–desorption isotherms, TGA and XRD techniques and further
2
the catalytic activity of this catalyst was studied in the synthesis of tetrazoles. All products were
obtained in good turnover numbers (TON), turnover frequency (TOF) and yields. The biological
activity of obtained tetrazoles was screened against several Gram-positive & Gram-negative
strains which these compounds showed significant antibacterial activity against of both Gram
negative and Gram positive bacteria. Also this catalyst can be recovered and recycled for several
times without any significant loss of its activity and stability of catalyst after recycling was
confirmed by SEM, FT-IR, AAS and N adsorption–desorption isotherms. Heterogeneity nature
2
of Cu-TBA@biochar as catalyst was studied by recycling of this catalyst and hot filtration test.
Acknowledgment
Authors thank the research facilities of Ilam University, Ilam, Iran, for financial support of this
research project.
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2
7

Graphical Abstract
Fabricated copper catalyst on biochar nanoparticles for the synthesis of
tetrazoles as antimicrobial agents
*
Parisa Moradi, Maryam Hajjami , Bahman Tahmasbi
CN
Cu-TBA@biochar
PEG-400
N
N
N
Cu
N
+ NaN3
S
R
N
H
R
N
16 Sample
Si
O
Antimicrobial active
compounds
N
O
Cu
O
N
S
O
Si
O
Biochar
O
Cu-TBA@biochar
In this work, biochar nanoparticles were prepared from pyrolysis of chicken manure and further
a new copper catalyst was supported on its surface (Cu-ABA@biochar) for the synthesis of
antimicrobial derivatives of tetrazole in high TOF and TON values. The biological activity of
obtained tetrazoles was screened against several Gram-positive & Gram-negative bacteria
strains. The structure of this catalyst has been characterized by SEM, EDS, WDX, XRD, TGA,
N adsorption–desorption isotherms, FT-IR and AAS techniques.
2
2
8